Accurate measurements of cellular mechanical properties are critical to understand a cell's biological response to its environment. The quality of mechanical measurements depends greatly on the attachment method of the apparatus to the cell specimen. The attachment method must provide a strong, reliable connection without altering the integrity of the specimen. Multi-cellular specimens can be attached easily because the exterior cells of the specimen chain can be attached to without fear of damaging the integrity of the interior cells. However, multi-cellular testing presents additional complications, including: 1) heterogeneous electrical activity and stress/strain distributions; 2) the influence of the extracellular matrix on both active and passive properties; and 3) non-uniform orientation of each cell. (Sugiura, Nishimura, Yasuda, Hosoya, and Katoh, “Carbon fiber technique for the investigation of single-cell mechanics in intact cardiac myocytes”, Natural Protocols 2006; 1: 1453, citing Brady A J, “Mechanical properties of isolated cardiac myocytes”, Physiol. Rev. 1991; 71: 413-28 and Gamier D, “Attachment procedures for mechanical manipulation of isolated cardiac myocytes: a challenge”, Cardiovasc Res. 1994; 28: 1958-64). For these reasons, the most accurate measurements can be obtained from single-cell specimens. Single cells do not have any attachment points (e.g., tendons, connective tissue), as with multi-cellular preparations (whole muscles or muscle strip preparations).
Attachment methods for single-cell specimens are much more tedious than those used on multi-cell specimens. There are no available external attachment locations forcing the apparatus to attach directly to the cell membrane. Therefore, the attachment method must not disturb the specimen while still providing strong enough attachment strength to allow for the application of large forces to the specimen. There have been a number of proposed attachment methods including wrapping, glass micro-needles, and suction micropipettes. (Nishimura, Seo, Nagasaki, Hosoya, Yamashita, Fujita, Nagai, and Suigiura, “Responses of single-ventricular myocytes to dynamic axial stretching”, Biophys and Mol Bio. 2008; 97: 282-97.) Each of these methods is difficult to reproduce and requires extensive technical expertise.
Another known attachment technique uses carbon fibers. Attachment is achieved by gently pressing the tip of the carbon fibers against the cell membrane. (Sugiura et al., citing Brady and Gamier.) This method is also undesirable because the carbon fiber's ability to stick to ventricular myocytes is inconsistent. Also, the method does not provide a reliable attachment point for cellular contractions greater than 2.42 μN, which is less than the force required to produce large cellular contractions. (Yasuda, Sugiura, Kobayakawa, Fujita, Yamashita, Katoh, Saeki, Kaneko, Suda, Nagai, Sugi, “A novel method to study contraction characteristics of a single cardiac myocytes using carbon fibers”, Am. J. Physiol. Heart Circ Physiol. 2001; 281: 3: H1442-H1446.) Moreover, the force measurements are dependent upon calculations of the carbon fibers' shortening and compliance which introduces unnecessary variables into the measurement calculations. (Nishimura et al.)
Adhesives have also been used to attach single-cell specimens. Silicon, poly-1-lysine, “Great Stuff” by Dow Chemical, and cyanoacrylate glue are known to have been used. Each of the identified compositions are limiting because a relatively large area of the cell membrane must be glued to achieve proper attachment. In addition, preparation time is extended because the adhesives require an extended amount of time to set and some can only be used on skinned myocytes. (Suigiura et al and Bluhm, McCulloch, and Lew, “Active force in rabbit ventricular myocytes”, J. Bomech. 1995; 28: 1119-1122.) While cyanoacrylate glue sets quickly and provides a strong grip, exposure will kill the cell in a short period of time, leaving a small window of opportunity to test the specimen.
Other adhesives are available that are specifically marketed for cell adhesion. These include ECM gel from Sigma-Aldrich, Inc. (Sigma-Aldrich, Inc. ECM Gel Product Information Sheet, www.sigma-aldrich.com), Matrigel™ from BD Biosciences, Inc. (BD Matrigel™ Basement Membrane Matrix Product Description, www.bdbiosciences.com), and Cell-Tak™ from BD Biosciences, Inc. (BD Cell-Tak™ Cell and Tissue Adhesive Product Manual. 1991, www.bdbiosciences.com). Matrigel™ and the ECM gel from Sigma-Aldrich are extra-cellular matrices (“ECMs”) derived from Engelbreth-Holm-Swarm mouse sarcoma. (BD Matrigel™ Basement Membrane Matrix Product Description, www.bdbiosciences.com). Both are too viscous and allow for specimen movement during mechanical testing. Cell-Tak™ is a bioadhesive derived from the polyphenolic proteins of marine mussels. (BD Cell-Tak™ Cell and Tissue Adhesive Product Manual. 1991, www.bdbiosciences.com) It has been used to attach single-cells extracted from the patellar tendon of matured Japanese white rabbits. (Miyazaki, Hasegawa, Hayashi, “A newly designed tensile tester for cells and its application to fibroblasts”, J. Biomech. 2000; 33: 97-104) However, during these tests there was approximately a 40% failure rate for the Cell-Tak™ bond. (Miyazaki et al.) Further, no test using the Cell-Tak™ adhesive exceeded a maximum load of 1.1 μN, which is much less than the load required for testing on myocytes. (Miyazaki et al.)